Cape-loaded targeted microvesicular cancer drug and method for developing the same

ABSTRACT

A method of producing a caffeic acid phenethyl ester loaded microvesicular cancer drug targeting SH-SY5Y neuroblastoma cancer. The method includes seeding a plurality of skin stem cells in a medium; enabling the plurality of skin stem cells in the medium to reach a confluency of 70-80% to be seeded in a plurality of culture plates; preparing a caffeic acid phenethyl ester stock solution by dissolving the caffeic acid phenethyl ester in dimethyl sulfoxide DMSO; performing a microvesicle isolation from specialized skin cells; adding the caffeic acid phenethyl ester into a plurality of microvesicles; and separating the caffeic acid phenethyl ester loaded microvesicles from the caffeic acid phenethyl ester which is free in the solution and not loaded to a microvesicle.

CROSS-REFERENCE TO THE RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 16/955,087, filed on Jun. 18, 2020, which is the national stageentry of International Application No. PCT/TR2018/050816, filed on Dec.17, 2018, which is based upon and claims priority to Turkish PatentApplication No. 2017/20642, filed on Dec. 18, 2017, the entire contentsof which are incorporated herein by reference.

TECHNICAL FIELD

The present invention relates to production of a drug which is specificfor SH-SY5Y neuroblastoma cancer by loading CAPE to microvesicles thatthe specialized skin cell leaves into the medium.

BACKGROUND

CAPE (caffeic acid phenethyl ester) is one of the most widely studiedactive components of poplar type propolis. CAPE has antioxidant,antineoplastic, antitumoral and cytoprotective effects. CAPE inhibitscarcinogenesis, cell cycle and metastasis, and induces apoptosis [1].One of the interesting and important effects of CAPE is that when usedwith various antibiotics (streptomycin, vancomycin, isoniazid,ethambutol) and cancer drugs (mitomycin, doxorubicin, cisplatin,methotrexate); it reduces the toxicity occurring dependent on the saiddrugs [2].

It has been shown that CAPE (40 μM) dose-dependently stops cellproliferation, induces cell cycle arrest and apoptosis, and suppressesangiogenesis in estrogen receptor positive (ER+) MCF-7, estrogenreceptor negative (ER−) MDA-MB-231, and triple negative (ER−, PR−,HER2−) TNBC breast cancer cell lines [3]. Additionally, it has also beendetermined that CAPE (40 μM) inhibits proliferation of breast cancerstem cells [4]. It has been shown that CAPE dose-dependently inhibitscell proliferation in LNCaP, DU-145 and PC-3 prostate cancer cell linesand Akt signaling pathway [5]. It has been demonstrated that CAPE (50μM) prevents proliferation by inducing S and G2/M phase cell cyclearrests and induces apoptosis in the cells in ME180 cervical cancer celllines [6]. It was determined that CAPE (100 μM) treatment decreased G1phase cell population, increased G2/M phase cell population and inducedapoptosis in the cells by inhibiting the Akt signaling pathway in TW2.6oral squamous cell carcinoma cell line [7]. It has been determined thatin the SK-1 Hep1 liver cancer cell line, to which combined treatment ofCAPE (30 μg/ml) and TRAIL was applied, caspase activation increased,cell death receptors were activated via JNK and p38 signaling pathwaysand apoptosis was induced in the cells [8]. In the experimental studies,it was observed that CAPE inhibited the toxicity occurring duringchemotherapy and radiotherapy. It is reported that the prophylacticadministration of CAPE prevented the damage caused by doxorubicin in thetissues of kidney [9], heart [10] and brain [11], and the liver damagedepending on administration of cisplatin [12] and tamoxifen [13] inrats. It has also been shown that CAPE treatment decreased radiationinduced lung injury in rats [14]. In the study conducted with CAPE inSH-SY5Y neuroblastoma cell line, CAPE was applied in the range of 4-20μM, but no effect was observed in the SH-SY5Y cells [15].

In another study, CAPE (100 μg/ml) was loaded in methoxy poly(ethyleneglycol)-b-poly(ε-caprolactone) (CE) copolymer and it was applied in CT26colon carcinoma cell line. When the results were compared withunaccompanied application of CAPE, no difference was observed on theeffects thereof on cell proliferation and death [16].

The problems encountered in the state-of-the-art applications can belisted as follows:

-   -   Not being specific to the cell and tissue.    -   Requirement to be used at high concentrations.    -   Activation of mononuclear phagocyte system.    -   Cancerous tissues showing chemical resistance in drug        applications.

SUMMARY

The objective of the present invention is to provide a cell-specifictargeted vesicle development by loading CAPE to the microvesiclesobtained as a result of the culturing and subsequent specialization(differentiation) of the tissue cells.

Another objective of the invention is to use CAPE at lowerconcentrations compared to the state-of-the-art applications to minimizeits toxicity to both the cells other than the targeted cells and thebody.

BRIEF DESCRIPTION OF THE DRAWINGS

“Development of a CAPE loaded microvesicular cancer drug targetingSH-SY5Y neuroblastoma cancer” developed to fulfill the objectives of thepresent invention is illustrated in the accompanying figures, in which:

FIG. 1 is a graphical representation of the combinations formed byloading CAPE to the microvesicles obtained from SH-SY5Y, PC3 and PNT-1cells on cell viability of SH-SY5Y cells in an application of 48 hours.(CAPE: Caffeic Acid Phenethyl Ester, MV: Microvesicle, *: P<0.05)

FIG. 2 is a graphical representation of the effect of the combinationsobtained by loading different doses of CAPE (caffeic acid phenethylester) to the microvesicles obtained from the cells, which aredifferentiated from stem cells into healthy nerve cells, on cellviability of SH-SY5Y cells in an application of 48 hours. (CAPE: CaffeicAcid Phenethyl Ester, MV: Microvesicle, *: P<0.05)

DETAILED DESCRIPTION OF THE EMBODIMENTS

The invention is a cancer drug obtained by loading drug (CAPE) into themicrovesicles produced by the differentiated skin stem cells, and it isapplied due to the cytotoxic effect of these cellular vesicles on cancercells. The drug is loaded to the vesicles of the cells, which arespecialized by being treated with growth factors applied to providenerve cell properties. Within the scope of the invention, the cells;which are differentiated by administering a Neurobasal solutionpreferably containing 10 ng/ml bFGF, 10 ng/ml EGF, 1% B7 supplement, 1%ITS (insulin, transferrin and selenium), 10% Glutamine and 1% PSA for12-14 days; are provided with new properties by this method renderingthe vesicles to target the SHSY5Y cancer cells. By means of theinvention, the cellular vesicles are loaded with CAPE thereby acquiringthe feature of specifically recognizing the SHSY5Y cancer cells.

There are two embodiments within the scope of the invention. In thefirst embodiment; by means of drug (CAPE (caffeic acid phenethyl ester)loading, cytotoxic property is provided to the cellular vesicles whichare produced by changing the factors such as the temperature of 37° C.,pH, 5% carbon dioxide and the DMEM, F12, RPMI media that enable cellgrowth, in addition to the growth conditions of the cells (stem cells,cell lines, primer cells, cancer cells, cells obtained from tissues). Inthe second embodiment; by means of drug (CAPE (caffeic acid phenethylester) loading, cytotoxic property is provided to the cellular vesicleswhich are produced by treating the cells (stem cells, cell lines, primercells, cancer cells, cells obtained from tissues) with other chemicals(growth factors such as bFGF, EGF, NGF; hormones such as melatonin,insulin, lactoferrin; vitamins such as ascorbic acid, folic acid; andminerals such as calcium, magnesium, boron) in addition to the cells'own growth conditions. It has been observed that these vesicles, whichare collected from the stem cells differentiated into nerve cells, whiledemonstrating a nerve cell recognition feature, they also exhibitcytotoxicity specific to SHSY5Y after treatment for 12-14 days as seenin FIG. 1. The term “specialized” is given as a result of the comparisonof the SHSY5Y cell with the other cells as seen in FIG. 1.

In one embodiment of the invention, the microvesicles; which areproduced by the cells specific to a particular cancer type by means ofthe differentiation of the cells to the tissue cells, where the saidtumor formation is observed, acquired on the 12-14th days rather than25-30 days; are used. Accordingly, the microvesicles produced by thecells are used because the specific characteristics of SHSY5Y cells,which are acquired by differentiation of the stem cells subjected tonerve cell differentiation, are carried in the early stage of nervedifferentiation by differentiation at 12-14 days rather than 25-30 days.

In one embodiment of the invention, with the purpose of developing aCAPE loaded microvesicular cancer drug targeting SH-SY5Y neuroblastomacancer, CAPE loading at a concentration of 5 μM to 100 μM is performedto the cellular microvesicles produced via the nerve cells; and thesevalues are far more advantageous over the toxic high amounts of >100 μMapplied in the previous individual CAPE applications in the literature.

The cancer drug product, which is produced by the CAPE loadedspecialized cellular microvesicles obtained in the scope of theinvention, has no toxic effect on healthy cells and other cell lines,while showing toxic effect only on a specific type of cancer. As shownin FIG. 1, and as mentioned above, the specialized microvesicles showcytotoxic effect only on SHSY5Y cells.

The inventive development of CAPE loaded microvesicular cancer drugtargeting SH-SY5Y neuroblastoma cancer comprises the steps of

-   -   Seeding (culturing) skin stem cells in a medium,    -   Implementing the specialization protocol, which enables the        cells in a culture medium reaching sufficient confluency        (70-80%) to be seeded in 6-well cell culture plates whereby the        feature of neuroblastoma recognition is provided to the cells        enabling them to become targeted,    -   Dissolving CAPE (caffeic acid phenethyl ester) in DMSO (Dimethyl        sulfoxide) thereby preparing the stock solution (thus different        concentrations can be obtained from the more concentrated stock        solution),    -   Performing microvesicle isolation from the specialized skin        cells,    -   Adding CAPE into the microvesicles,    -   Separating the CAPE loaded microvesicles from the CAPE which is        free in the solution and not loaded to a microvesicle,    -   Obtaining the CAPE loaded microvesicular cancer drug targeting        SH-SY5Y neuroblastoma cancer which is the final product.

In the preferred embodiment of the invention, during the step ofcarrying out the culturing of the skin stem cells; the skin stem cell,SH-SY5Y, PNT-1A, PC-3 cells are cultured preferably in DMEM (Dulbecco'sModified Eagle's Medium) medium containing 10% fetal bovine serum and 1%PSA in cell culture incubators preferably at 37° C. and 5% CO₂.

In the preferred embodiment of the invention, during the step ofspecializing the cells in the culture medium, a Neurobasal solutionpreferably containing 10 ng/ml bFGF, 10 ng/ml EGF, 1% B7 supplement, 1%ITS (insulin, transferrin and selenium), 10% Glutamine and 1% PSA isprepared and then the specialization protocol is applied byadministering this specialization solution to the cells seeded in 6-wellcell culture plates once in two days for 12 to 14 days.

In the preferred embodiment of the invention, in the step of preparingthe stock solution, 45.75 mg of CAPE is preferably dissolved in 3.22 mLof DMSO (Dimethyl sulfoxide). The final concentration obtained isapproximately 50,000 μM.

In the preferred embodiment of the invention, in the step ofmicrovesicle isolation from the specialized skin cells, the solutioncollected from the culture medium is centrifuged at 300 g for 10 minutesto remove the waste cells. The supernatant remaining at the upper partof the tube after the centrifugation is transferred to a new tube and itis centrifuged at 14,000 g for 30 minutes in order to remove possiblecell components. The supernatant remaining at the upper part of the tubeafter the centrifugation is transferred to a new tube and ½ volume ofthe kit buffer solution (solution A) is added, and it is incubated forone day at +4 degrees. The next day, after it is centrifuged at 16,000 gfor 1 hour, the pellet is dissolved in distilled water (dH₂O).

In the preferred embodiment of the invention, loading CAPE into themicrovescular structure is carried out by incubation at roomtemperature. Microvesicle solution is added to a 50 μM CAPE solutionpreferably prepared in 2 ml of PBS such that the final concentrationwill be 100 μg/ml, and the mixture is incubated for 20 minutes at roomtemperature (25° C.). Then, the precipitation process is carried outusing the isolation kit to obtain the substance-loaded vesicles. Theresulting substance-loaded pellet is dissolved in distilled water(dH₂O).

In the “CAPE loaded microvascular cancer drug targeting SHSY5Yneuroblastoma cancer (Micro-CAPE)” of the present invention, the skinstem cells, PNT-1A, PC-3 and SH-SY5Y cells are used. The toxicity of thetargeted microvesicles, which are obtained by specialization of the stemcells, to the SH-SY5Y cells was observed. By loading CAPE to themicrovesicles obtained in this study, a cell-specific targeted vesicleis developed while the toxicity to both the other cells and the body isminimized by using much lower concentrations of CAPE compared to theconcentrations previously used in the literature.

Within the scope of the invention, first of all, the skin stem cells,SH-SY5Y, PNT-1A and PC-3 cells were cultured in Dulbecco's modifiedEagle's medium (DMEM) containing 10% fetal bovine serum (Invitrogen) and1% PSA (Biological Industries, Beit Haemek, Israel) at a temperature of37° C. in cell culture incubators with 5% CO₂. The cells in the culturesolution reaching sufficient confluence (70-80%) are seeded in 6-wellculture plates and the specialization protocol is applied thereon for 13days with Neurobasal solution containing 10 ng/ml bFGF, 10 ng/ml EGF, 1%B7 supplement, 1% ITS (insulin, transferrin, and selenium), 10%Glutamine and 1% PSA by changing the media every other day. In themeantime, the process of preparing the stock solution is started. Forthis purpose, 45.75 mg of CAPE is dissolved in 3.22 mL of DMSO (Dimethylsulfoxide). The final concentration obtained is 50,000 μM.

Microvesicles are isolated from the skin cells on which specializationprotocol is applied. EX01 Exo-spin™ kit was used for microvesicleisolation from the specialized skin cells in the scope of the invention.The medium collected from the culture medium is centrifuged at 300 g for10 minutes in order to remove the waste cells. The supernatant istransferred to a new tube, and it is centrifuged at 14,000 g for 30minutes in order to remove possible cell components. The supernatantobtained by this centrifugation is transferred to a new tube and ½volume of the kit buffer solution (solution A) is added, and it isincubated for one day at +4 degrees. The next day, after it iscentrifuged at 16,000 g for 1 hour, the pellet is dissolved in distilledwater (dH₂O).

Loading of CAPE, which is prepared as a separate solution, into themicrovesicles is carried out at room temperature. To a solution of 50 μMCAPE prepared in 2 ml PBS is added the microvesicle solution such thatthe final concentration will be 100 ug/ml. The mixture is incubated for20 minutes at room temperature (25° C.) and then, the precipitationprocess is carried out using the isolation kit to obtain CAPE loadedvesicles. The resulting substance-loaded pellet is dissolved indistilled water (dH₂O).

By means of the inventive “Development of CAPE loaded microvesicularcancer drug targeting SHSY5Y neuroblastoma cancer (Micro-CAPE)”, thedrug is enabled

-   -   to be used at much lower concentrations (5 μM),    -   to be specific to cell type,    -   to pass the blood-brain barrier,    -   not to cause inflammation,    -   not to have any toxicity to the body,    -   to prevent resistance of the cancer type,    -   to be metabolized in the cell after use,    -   to remain in the circulation for a long time.

Experimental Studies

Measuring the Amount of CAPE that is Loaded

After the loading process, measurement of the amount of CAPE transferredinto the microvesicular structure was performed based on thespectrophotometric measurement method. When measuring the amount of CAPEthat was loaded, the intrinsic radiation of the molecule at 323 nmwavelength was utilized. Different concentrations (1-100 μM) of CAPEwere measured at the wavelength of 323 nm to form a standard curve. Theamount of CAPE that was loaded was determined using two interrelatedmethods. Firstly, the amount of CAPE that was loaded was determined bymeasuring the amount remaining in the supernatant after precipitation ofthe CAPE-loaded microvesicles. Secondly, it was determined byfractionation of the membrane structures of the microvesicles loadedwith the precipitated substance, and measurement of the amount of CAPEloaded to the vesicular structure.

Determining Toxicity

After the cells were seeded in 96-well culture plates (CorningGlasswork, Corning, N.Y.) at 5000 cells/well in Dulbecco's modifiedEagle's medium (DMEM) containing 10% fetal bovine serum (Invitrogen) and1% PSA (Biological Industries, Beit Haemek, Israel) in the culturesolution, the viability levels of the cells were measured on day 1, 2and 3. Cell viability was determined by using3-(4,5-di-methyl-thiazol-2-yl)-5-(3-carboxy-methoxy-phenethyl)-2-(4-sulfo-phenethyl)-2H-tetrazolium(MTS)-method (CellTiter96 AqueousOne Solution; Promega, Southampton,UK). 10 μl MTS solution was added onto the cells within a 100 μl mediumand it was incubated at 37° C. in dark for 2 hours. After the incubationprocess, cell viability was observed by performing absorbancemeasurement via ELISA plate reader (Biotek, Winooski, Vt.) device at 490nm wavelength.

REFERENCES

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What is claimed is: 1.-6. (canceled)
 7. A method of producing a caffeicacid phenethyl ester (CAPE) loaded microvesicular cancer drug targetingSH-SY5Y neuroblastoma cancer, comprising the steps of: 1) seeding aplurality of skin stem cells in a culture medium, 2) implementing aspecialization protocol to obtain a plurality of specialized skin cells,wherein the specialization protocol enables the plurality of skin stemcells in the culture medium reaching a confluency of 70-80% to be seededin a plurality of 6-well cell culture plates whereby a feature ofneuroblastoma recognition is provided to the plurality of skin stemcells enabling the plurality of skin stem cells to become targeted, 3)preparing a CAPE stock solution by dissolving the CAPE in dimethylsulfoxide (DMSO), 4) performing a microvesicle isolation from theplurality of specialized skin cells to obtain a plurality ofmicrovesicles, 5) adding the CAPE into the plurality of microvesicles,6) separating a plurality of CAPE loaded microvesicles from the CAPEthat is free in the CAPE stock solution and not loaded to the pluralityof microvesicles, 7) obtaining the CAPE loaded microvesicular cancerdrug targeting SH-SY5Y neuroblastoma cancer as a final product.
 8. Themethod according to claim 7, wherein during step 1, the plurality ofskin stem cells are cultured with SH-SY5Y cells, PNT-1A cells, PC-3cells in DMEM containing 10% fetal bovine serum, and 1% PSA in aplurality of cell culture incubators at a temperature of 37° C. and 5%CO₂.
 9. The method according to claim 7, wherein step 2 comprises:preparing a specialization solution, wherein the specialization solutionis a Neurobasal solution containing 10 ng/ml bFGF, 10 ng/ml EGF, 1% B7supplement, 1% insulin, transferrin, and selenium (ITS), 10% Glutamineand 1% PSA, and implementing the specialization protocol byadministering the specialization solution to the plurality of skin stemcells seeded in the plurality of 6-well cell culture plates once everytwo days for 12 to 14 days.
 10. The method according to claim 7, whereinstep 3 comprises preparing the CAPE stock solution with a finalconcentration of approximately 50,000 μM by dissolving 45.75 mg of theCAPE in 3.22 mL of DMSO.
 11. The method according to claim 7, whereinstep 4 comprises a first centrifugation, wherein a solution collectedfrom the culture medium is centrifuged at 300 g for 10 minutes to removea plurality of waste cells.
 12. The method according to claim 11,wherein step 4 further comprises a second centrifugation, wherein asupernatant remaining at an upper part of a centrifuge tube after thefirst centrifugation is transferred to a new centrifuge tube, and thesupernatant remaining is centrifuged at 14,000 g for 30 minutes in orderto remove a plurality of possible cell components.
 13. The methodaccording to claim 11, wherein step 4 further comprises an incubation,wherein a supernatant remaining at an upper part of a tube after thefirst centrifugation is transferred to a new tube and ½ volume of abuffer solution is added, and the supernatant remaining is incubated forone day at +4 degrees Celsius.
 14. The method according to claim 13,wherein following 1 day of the incubation, after the supernatantremaining is centrifuged at 16,000 g for 1 hour, and a pellet isdissolved in distilled water.
 15. The method according to claim 7,wherein in step 5, the CAPE loading into the plurality of microvesiclesis carried out by incubation at room temperature.
 16. The methodaccording to claim 15, wherein in step 5 comprises preparing a 50 μMCAPE solution in 2 mL PBS, and adding a solution of the plurality ofmicrovesicles to the 50 μM CAPE solution prepared in 2 ml of PBS toobtain a mixture such that a final concentration of the mixture is 100μg/ml, and then incubating the mixture for 20 minutes at the roomtemperature.
 17. The method according to claim 16, wherein step 6comprises performing a precipitation to the mixture incubated in step 5to obtain the plurality of CAPE loaded microvesicles, and step 7comprises dissolving the plurality of CAPE loaded microvesicles indistilled water.
 18. (canceled)